Neuroplasticity with alcohol dependence

酒精依赖的神经可塑性

• 批准号: 8373394
• 负责人: Scott C Steffensen
• 金额: $ 29.98万
• 依托单位: BRIGHAM YOUNG UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8373394
• 关键词: Affinity; Alcohol consumption; Alcohol dependence; Alcoholism; Alcohols; Area; Behavior; Behavioral; Brain; Brain-Derived Neurotrophic Factor; Cells; Charge; Chronic; Clinical; Complement; Correlative Study; Dependence; Diet; Disease; Dopamine; Dose; Electrophysiology (science); Ethanol; Ethanol dependence; Exposure to; GABA-A Receptor; Gene Expression; Glutamates; Goals; Homeostasis; Image; Imaging Techniques; Individual; Injection of therapeutic agent; Interneurons; Intervention; Liquid substance; Mediating; Midbrain structure; Molecular; Mus; Neuronal Plasticity; Neurons; Nucleus Accumbens; Opiate Addiction; Opioid; Pathway interactions; Pharmaceutical Preparations; Physiologic pulse; Physiological; Play; Population; Procedures; Property; Protein Tyrosine Kinase; Quantitative Reverse Transcriptase PCR; Receptor Activation; Relapse; Reporting; Research; Rewards; Role; Self Administration; Sensory; Slice; Small Interfering RNA; Societies; Structure; Synapses; Synaptic Transmission; System; Testing; Therapeutic Agents; Transcript; Tyrosine 3-Monooxygenase; Ventral Tegmental Area; Withdrawal; Work; alcohol exposure; alcohol reward; alcohol seeking behavior; base; clomeleon; dopaminergic neuron; evidence base; gamma-Aminobutyric Acid; indexing; insight; mesolimbic system; multidisciplinary; neurotransmission; novel; patch clamp; pre-clinical; programs; receptor; receptor expression; relating to nervous system; response; transmission process

DESCRIPTION (provided by applicant): The prevailing view is that enhancement of dopamine transmission in the mesocorticolimbic system underlies the rewarding properties of alcohol. This system consists of dopamine neurons in the midbrain ventral tegmental area (VTA) that innervate the nucleus accumbens and other limbic structures. Dopamine neurotransmission is controlled by local-circuit GABA interneurons. We have identified a homogeneous population of GABA neurons in the VTA that are excited by low-dose ethanol, but inhibited by moderate to high-dose ethanol, become tolerant to chronic ethanol, evince hyperexcitabililty during withdrawal, and accelerate in anticipation of ethanol self-reward. These correlative studies are complemented by our recent studies demonstrating a causal role for VTA GABA neurons in ethanol self-administration. VTA GABA neurons are widely accepted to be critical regulators of DA neurotransmission, but they may serve as unique and independent integrators of convergent information from sensory, cortical and limbic areas subserving alcohol reward and dependence. The core thesis underlying this proposal is that repeated exposure to alcohol causes adaptive changes in GABA(A) receptor [GABA(A)R]-mediated inhibition of VTA GABA neurons and contributes to the dysregulation of mesolimbic DA homeostasis that accompanies alcohol dependence (Gilpin and Koob, 2008; Koob and Le Moal, 1997). Based on previous work and preliminary results, we hypothesize that adaptation of VTA GABA neurons to chronic ethanol exposure and accompanying dependence results from a molecular switch in GABA(A)Rs on VTA GABA neurons, similar to what has been reported in our studies of opiate dependence (Laviolette et al., 2004; Vargas-Perez et al., 2009). We will employ multidisciplinary behavioral, electrophysiological, molecular and novel fluorescent imaging approaches to evaluate the adaptive effects of short-term and long-term ethanol exposure on GABA(A)R-mediated inhibition and glutamate (GLU) NMDAR-mediated excitation, and receptor expression, as well as the role of brain-derived neurotrophic factor (BDNF) tyrosine kinase B (TrkB) receptors in mediating the functional switch of GABA(A)Rs during ethanol dependence. Our studies will test the following hypotheses: 1) Withdrawal from a single exposure to ethanol (non-dependent condition) will enhance NMDAR-mediated GLU excitation of VTA GABA neurons, while withdrawal from chronic ethanol (dependent condition) will reduce GABA(A)R-mediated inhibition of VTA GABA neurons; 2) Withdrawal from chronic ethanol exposure will modify the expression of GABA(A)Rs; and 3) Withdrawal from chronic exposure to ethanol will result in a functional switch in GABA(A)R-mediated inhibition of VTA GABA neurons that is mediated by BDNF TrkB receptor activation. To test these hypotheses, we propose three Specific Aims in GAD GFP mice, wherein GABA neurons can be identified and characterized unambiguously. We will focus on mechanistic approaches in order to characterize the synaptic substrates in VTA GABA neurons that adapt in response to a single exposure (short-term) or multiple exposures to ethanol (long-term), and the role of BDNF and its high- affinity TrkB receptor in mediating the long-term adaptation of GABA(A)Rs. To test these hypotheses, we propose three Specific Aims in GAD GFP mice, wherein GABA neurons can be identified and characterized electrophysiologically: 1) We will evaluate spontaneous and evoked inhibitory and excitatory synaptic transmission, paired-pulse responses, total charge transfer, AMPAR/NMDAR ratio and AMPA rectification index using patch clamp electrophysiology. These studies will be accomplished by recording IPSCs and EPSCs from brain slices during withdrawal from a single injection of ethanol administered to mice 24 hrs previous (non-dependent condition) or in mice consuming ethanol in the forced liquid ethanol diet procedure (dependent condition); 2) We will evaluate the GABA(A)R subunit, NMDAR subunit, tyrosine hydroxylase, Cx36, and TrkB receptor transcript expression in VTA GABA neurons using single-cell quantitative RTPCR; and 3) We will evaluate the effects of BDNF TrkB receptor antagonists and TrkB depletion with siRNA TrkB on GABA(A)R-mediated inhibitory and NMDAR-mediated excitatory synaptic responses as in Aim 1. In addition, we will evaluate the hypothetical switch in GABA(A)R using the perforated patch procedure for individual VTA GABA neurons and using the novel Clomeleon fluorescent imaging procedure for populations of VTA GABA neurons. The proposed studies will provide important new insights into the role of GABA(A)Rs on VTA GABA neurons in alcohol dependence. VTA GABA neurons evince neuroadaptive responses in association with opiate dependence, characterized by a switch in functionality from being hyperpolarized by GABA to being depolarized. This switch appears to involve BDNF, as it triggers long-term changes in the functionality of GABA(A) receptors and a state of dependence without chronic opioids. We anticipate that the studies we propose will provide important new insights into the contributory role of VTA GABA neurons and their functional connectivity in ethanol consumption and the role of BDNF in mediating a switch in the functionality of GABA(A) receptors on VTA GABA neurons with alcohol dependence. Results from this study could provide a preclinical pharmacologic rationale for considering drugs that act selectively on GABA(A) receptor subtypes or on BDNF TrkB receptors as putative therapeutic agents for the treatment of alcohol dependence. PUBLIC HEALTH RELEVANCE: Alcoholism is a chronic relapsing disorder that has enormous impact on society. A major goal of research on alcoholism is to characterize the critical neural substrates that are most sensitive to alcohol, adapt in association with chronic alcohol and drive subsequent alcohol-seeking behavior. Currently, there are no evidence-based, clinically useful, pharmacotherapeutic interventions that might reverse the neuroadaptive effects of alcohol dependence. The long-term objective of our research program is to advance our understanding of the neural basis of alcohol reward and dependence and, subsequently, to identify therapeutic agents whose mechanisms of action would be predicted to have clinical utility in treating it. A population of GABA neurons in the midbrain that regulate dopamine neurons in the reward pathway appear to be promising candidates, as they are especially sensitive to alcohol, their activity correlates with rewarding behaviors and recent evidence indicates that they play a causal role in alcohol self-administration. Most importantly, they show remarkable adaptation to alcohol dependence. Thus, elucidating the molecular substrates that underlie this adaptation may suggest treatments to reverse alcohol dependence.

描述（由申请人提供）：普遍的观点是，中皮质边缘系统中多巴胺传递的增强是酒精的有益特性的基础。该系统由中脑腹侧被盖区 (VTA) 的多巴胺神经元组成，这些神经元支配伏核和其他边缘结构。多巴胺神经传递由局部电路 GABA 中间神经元控制。我们已经确定了 VTA 中的同质 GABA 神经元群体，它们被低剂量乙醇兴奋，但被中剂量至高剂量乙醇抑制，对长期乙醇变得耐受，在戒断期间表现出过度兴奋性，并在预期乙醇自我奖励时加速。我们最近的研究证明了 VTA GABA 神经元在乙醇自我给药中的因果作用，对这些相关研究进行了补充。 VTA GABA 神经元被广泛认为是 DA 神经传递的关键调节器，但它们可能作为来自感觉、皮质和边缘区域的汇聚信息的独特且独立的整合器，促进酒精奖赏和依赖性。该提议的核心论点是，反复接触酒精会导致 GABA(A) 受体 [GABA(A)R] 介导的 VTA GABA 神经元抑制发生适应性变化，并导致伴随酒精依赖的中脑边缘 DA 稳态失调（Gilpin 和 Koob，2008；Koob 和 Le Moal，1997）。基于之前的工作和初步结果，我们假设 VTA GABA 神经元对慢性乙醇暴露的适应以及伴随的依赖性是由 VTA GABA 神经元上的 GABA(A)R 分子开关引起的，类似于我们在阿片依赖研究中报道的情况 (Laviolette et al., 2004; Vargas-Perez et al., 2009)。我们将采用多学科行为、电生理学、分子和新型荧光成像方法来评估短期和长期乙醇暴露对 GABA(A)R 介导的抑制和谷氨酸 (GLU) NMDAR 介导的兴奋和受体表达的适应性影响，以及脑源性神经营养因子 (BDNF) 酪氨酸激酶 B 的作用 (TrkB) 受体在乙醇依赖过程中介导 GABA(A)R 的功能开关。我们的研究将测试以下假设：1）戒断单次乙醇暴露（非依赖性条件）将增强 NMDAR 介导的 GLU 对 VTA GABA 神经元的兴奋，而戒断长期乙醇（依赖性条件）将减少 GABA(A)R 介导的 VTA GABA 神经元抑制； 2) 戒断长期乙醇暴露会改变GABA(A)Rs的表达； 3) 戒断长期接触乙醇将导致 GABA(A)R 介导的 VTA GABA 神经元抑制功能发生转变，而这种抑制是由 BDNF TrkB 受体激活介导的。为了测试这些假设，我们在 GAD GFP 小鼠中提出了三个特定目标，其中可以明确地识别和表征 GABA 神经元。我们将重点关注机制方法，以表征 VTA GABA 神经元中响应单次暴露（短期）或多次暴露乙醇（长期）的突触底物，以及 BDNF 及其高亲和力 TrkB 受体在介导 GABA(A)R 长期适应中的作用。为了测试这些假设，我们在 GAD GFP 小鼠中提出了三个特定目标，其中 GABA 神经元可以通过电生理学方法进行识别和表征：1）我们将使用膜片钳电生理学评估自发和诱发的抑制性和兴奋性突触传递、配对脉冲反应、总电荷转移、AMPAR/NMDAR 比率和 AMPA 整流指数。这些研究将通过记录 24 小时前给小鼠单次注射乙醇（非依赖条件）或在强制液体乙醇饮食程序中消耗乙醇（依赖条件）的小鼠退出期间脑切片中的 IPSC 和 EPSC 来完成； 2）我们将使用单细胞定量RTPCR评估VTA GABA神经元中GABA（A）R亚基、NMDAR亚基、酪氨酸羟化酶、Cx36和TrkB受体转录表达； 3) 我们将评估 BDNF TrkB 受体拮抗剂和 siRNA TrkB 消除 TrkB 对 GABA(A)R 介导的抑制性和 NMDAR 介导的兴奋性突触反应的影响，如目标 1 所示。此外，我们将使用单个 VTA GABA 神经元的穿孔贴片程序并使用新型 Clomeleon 评估 GABA(A)R 中的假设开关 VTA GABA 神经元群体的荧光成像程序。拟议的研究将为 GABA(A)Rs 对 VTA GABA 神经元在酒精依赖中的作用提供重要的新见解。 VTA GABA 神经元表现出与阿片依赖相关的神经适应性反应，其特征是功能从 GABA 超极化到去极化的转变。这种转变似乎涉及 BDNF，因为它会引发 GABA(A) 受体功能的长期变化，并导致无需长期阿片类药物的依赖状态。我们预计，我们提出的研究将为 VTA GABA 神经元的贡献作用及其在乙醇消耗中的功能连接以及 BDNF 在介导酒精依赖性 VTA GABA 神经元上 GABA(A) 受体功能转换中的作用提供重要的新见解。这项研究的结果可以为考虑选择性作用于 GABA(A) 受体亚型或 BDNF TrkB 受体的药物作为治疗酒精依赖的推定治疗药物提供临床前药理学原理。 公共卫生相关性：酗酒是一种慢性复发性疾病，对社会产生巨大影响。酗酒研究的一个主要目标是确定对酒精最敏感、与长期饮酒相关的适应并驱动随后的饮酒行为的关键神经基质的特征。目前，尚无基于证据的、临床上有用的药物治疗干预措施可以逆转酒精依赖的神经适应性影响。我们研究计划的长期目标是增进我们对酒精奖赏和依赖的神经基础的理解，并随后确定其作用机制预计在治疗中具有临床效用的治疗药物。中脑中调节奖励通路中多巴胺神经元的 GABA 神经元群似乎是有前途的候选者，因为它们对酒精特别敏感，它们的活动与奖励行为相关，最近的证据表明它们在酒精自我管理中发挥着因果作用。最重要的是，他们对酒精依赖表现出显着的适应能力。因此，阐明这种适应的分子底物可能会提出逆转酒精依赖的治疗方法。